Biochemical Systematics and Ecology 36 (2008) 638–645

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Biochemical Systematics and Ecology

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Phytochemical differentiation of Galanthus nivalis andGalanthus elwesii (Amaryllidaceae): A case study

Strahil Berkov a, Jaume Bastida a, Borijana Sidjimova b,Francesc Viladomat a, Carles Codina a,*

a Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmacia, Universitat de Barcelona,Av. Joan XXIII s/n, 08028 Barcelona, Catalonia, Spainb Department of Applied Botany, Institute of Botany, Bulgarian Academy of Sciences, 23 Acad. G. Bonchev Street, 1113-Sofia, Bulgaria

a r t i c l e i n f o

Article history:Received 7 August 2007Accepted 6 April 2008

Keywords:Galanthus nivalisGalanthus elwesiiAlkaloidsChemotaxonomyGC/MS

* Corresponding author. Tel.: þ34 934024493; faE-mail address: carlescodina@ub.edu (C. Codina)

0305-1978/$ – see front matter � 2008 Elsevier Ltddoi:10.1016/j.bse.2008.04.002

a b s t r a c t

The alkaloid patterns of two occasionally sympatric Galanthus nivalis and Galanthus elwesiipopulations were studied by GC/MS. Thirty-seven alkaloids were detected, 25 for G. nivalisand 17 for G. elwesii. Only five alkaloids were found to occur in both species. The popula-tions of Galanthus differed in their alkaloid biosynthetic pathways. Thus, the alkaloidpattern of G. nivalis was dominated by compounds coming from a para–para0 oxidativecoupling of O-methylnorbelladine. The predominant alkaloids in the roots of this specieswere found to belong to the lycorine and tazettine structural types; bulbs were dominatedby tazettine, leaves by lycorine and flowers by haemanthamine type alkaloids. In contrast,the alkaloid pattern of G. elwesii was dominated mainly by compounds coming from anortho–para0 oxidative coupling. The predominant alkaloids in G. elwesii roots, bulbs andleaves were those of homolycorine type, whereas the flowers accumulated mainly tyra-mine type compounds. The chemotaxonomical value of the alkaloids found in the studiedspecies is discussed.

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1. Introduction

Species of the genus Galanthus L. (Amaryllidaceae) are difficult to distinguish and classify because of the lack of clearlydefinable morphological characteristics. The search for other useful systematic information has produced little consensusto the enumeration of the species, divisions within the genus and relationships among their various components (Davisand Barnett, 1997). Beside morphological features, cariological (Kamari, 1981), anatomical (Davis and Barnett, 1997) andDNA (Zonneveld et al., 2003) methods have been used to clarify the taxonomy of the genus.

The alkaloids found in the plants of the family Amaryllidaceae possess interesting pharmacological properties such as ace-tylcholinesterase inhibitory activity, cytotoxity, antitumoral, etc. (Bastida et al., 2006). Seven of the about 18 species of thegenus Galanthus have been studied for their alkaloid composition. A brief overview on the alkaloid distribution among thestudied species shows that the genus is a rich source of new alkaloids (Latvala et al., 1995; Noyan et al., 1998; �€Unver, 2007;Berkov et al., 2007a), but there is no information about the organ distribution of these secondary metabolites in the genus.

The genus Galanthus is represented by two species in Bulgaria, Galanthus elwesii Hook. fil. and Galanthus nivalis L. (Kozuharov,1992;Petrovaetal.,1999).G.elwesiiexhibitsagreatmorplhologicalvariabilityandasaresult, ithasbeenassociatedwithanumber

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S. Berkov et al. / Biochemical Systematics and Ecology 36 (2008) 638–645 639

of other species that have glaucous leaves and two marks on the inner perianth segments, namely Galanthus gracilis �Celak.,Galanthus graecus Orph. ex Boiss., and Galanthus maximus Velen. (Davis, 1999). G. nivalis is easily distinguished by the singlemark on the inner perianth segments (Petrova et al.,1999). However, the taxonomical status of the genus Galanthus in Bulgariahaschangedseveraltimes.Upto1966,onlyG.nivaliss.l. includingseveralsubspecieshadbeenacceptedbythebotanists(Stojanovand Stefanov,1948; Jordanov,1964). Afterwards, it was divided into G. elwesii Hook. fil. and G. nivalis L. (Stojanov et al.,1966). G.graecusOrph.exBoiss.hadbeenalsoreportedtogrowinthatcountry(Delipavlov,1968,1971)butatpresenttheoccurrenceof thisspecies in Bulgaria is not confirmed.

There are few reports regarding the occurrence of alkaloids in G. nivalis, and the information is confusing owing to thetaxonomical changes in this species. Valkova (1961) and Bubeva-Ivanova and Pavlova (1965) had reported the occurrenceof six alkaloids in G. nivalis var gracilis L. (�Celak.) collected in Bulgaria. At present, however, the plants studied by those authorscannot be ascribed unambiguously to G. elwesii or G. nivalis due to the lack of voucher specimens, insufficient description ofthe collected plant material, and the different taxonomical features used for the determination of the Galanthus taxons inBulgaria. Kaya et al. (2004) found five alkaloids for G. nivalis L. subsp. silicicus (Baker) Guttl.-Tann., a taxon which havebeen reported as a synonym of Galanthus silicicus Baker by other authors (Davis and Barnett, 1997; Davis, 1999).Galanthamine, narwedine, nivalidine, lycorine, hippeastrine and tazettine have been isolated from G. nivalis as well (Briggset al., 1956; Kalashnikov, 1970). To our knowledge, about 30 alkaloids have been identified in G. elwesii (Boit and Horst,1955; Cherkasov, 1977; Latvala et al., 1995; Berkov et al., 2004, 2007b).

Up to the moment, no attempts to use secondary metabolites for chemotaxonomical studies have been done within thegenus Galanthus. In our previous work, relatively high biochemical diversity in G. elwesii has been revealed (Berkov et al.,2004). In a further search for natural populations of Galanthus plants, we found G. nivalis and G. elwesii occasionally sympatricin the north-east part of Bulgaria. Plant material for isolation of the characteristic alkaloids (Berkov et al., 2007a,b) and fora study on the alkaloid patterns in the different plant organs was gathered. In this work we report on the differences inthe alkaloid metabolism of these sympatric G. nivalis and G. elwesii populations. Extracts from plant organs of several indi-viduals were analyzed in order to obtain a general picture of the organ-to-organ alkaloid distribution. In addition, thevariations in the alkaloid profile within populations were studied by TLC, analyzing 50 individuals (leaf extracts) of eachpopulation.

2. Materials and methods

2.1. Plant material

Ten individuals of G. nivalis and 10 of G. elwesii were collected in flowering stage in March 2003 from sympatric popula-tions (plants of the two species coexisting in the same location) near the village of Obrochishte, district of Varna, Bulgaria. Thespecies were separated according to the accepted morphological difference between them: the presence in G. elwesii, andabsence in G. nivalis, of green marks on the top of the inner perianth segments (Petrova et al., 1999). Voucher specimenswere deposited at the Institute of Botany, Bulgarian Academy of Sciences, Sofia, and registered with numbers SOM 162922(G. nivalis) and SOM 162923 (G. elwesii).

2.2. Alkaloid extraction

Samples (fresh) from different plant organs were separately extracted with ethanol (3�100 ml, 72 h each). The solventwas evaporated under vacuum and the residues were dissolved in 30 ml of 2% sulfuric acid and defatted with diethylether(5� 40 ml). After that, the aqueous layers were basified with 25% ammonia to pH 9.5–10 and the alkaloids extracted withchloroform (3� 50 ml). After evaporation of the organic solvent, the dried extracts were dissolved in methanol (5 mg ofextract in 250 ml of methanol) for further GC/MS analysis.

2.3. GC/MS analysis

The capillary gas chromatography/mass spectrometry (GC/MS) analyses were recorded on a Hewlett–Packard 6890þMSD5975, (Hewlett–Packard, Palo Alto, CA, USA) operating in EI mode at 70 eV. A HP-5 MS column (30 m� 0.25 mm� 0.25 mm)was used. The temperature program was: 100–180 �C at 15 �C min�1, 180–300 at 5 �C min�1and 10 min hold at 300 �C. Injec-tor temperature was 250 �C. The flow rate of carrier gas (helium) was 0.8 ml min�1. Split ratio was 1:20. One microliter of thesolution was injected.

The alkaloids were identified applying co-chromatography with standards previously isolated in our laboratory from G.nivalis, G. elwesii and other amaryllidaceous plants, comparing the mass spectral fragmentation of the compounds withstandard reference spectra from NIST 05 database (NIST Mass Spectral Database, PC-Version 5.0 (2005), National Instituteof Standardization and Technology, Gaithersburg, MD), and comparing the obtained spectra with those reported in the liter-ature or closely related isolated compounds as indicated in Table 1. Mass spectra were deconvoluted using AMDIS 2.64software (NIST). Kovats retention indexes (RI) of the compounds were recorded with standard calibration n-hydrocarbonmixture (C9–C36) using AMDIS 2.64 software.

Table 1Alkaloids identified in G. nivalis (GN) and G. elwesii (GE) plant organs by GC/MS

Compound RI Mþ Rel. int. (%) Roots Bulbs Leaves Flowers

GN GE GN GE GN GE GN GE

Tyramine typeN-Methyltyramine (1)a,d 1454 151 (40) 135 (6), 120 (15), 107 (100), 91 (27), 77 (89),

65 (25), 55 (41)<0.1 <0.1 <0.1 <0.1

Hordenine (2)b-1 1463 165 (1) 120 (1), 107 (1), 91 (1), 77 (4),65 (1), 58 (100)

6.2 21.6 2.1 20.5 0.26 30.2 <0.1 58.9

Total 6.2 21.6 2.1 20.5 0.26 30.2 58.9

MiscellaneousIsmine (3)b-2 2280 257 (35) 238 (100), 211 (6), 196 (8), 168 (6), 154 (3),

106 (4), 77 (3)9.3 9.9 2.4 2.6

Trisphaeridine (4)c-1 2282 223 (100) 222 (38), 167 (8), 165 (9), 164 (14), 138 (20),137 (9), 111 (13)

<0.1 <0.1 <0.1 <0.1 <0.1

Total 9.3 9.9 2.4 2.6

Haemanthamine typeVittatine (5)b-3 2473 271 (100) 228 (25), 199 (95), 187 (85), 173 (27), 157 (22),

128 (32), 115 (33)<0.1 <0.1

Haemanthamine (6)b-3 2643 301 (13) 272 (100), 240 (16), 225 (6), 211 (13), 199 (7),181 (21), 153 (8)

<0.1 0.8 0.2

Hamayne (7)b-2 2700 287 (3) 258 (100), 242 (6), 211 (12), 186 (17), 181 (11),153 (10), 128 (19)

14.6 7.2

A-1 (8) 2988 *355 (86) 286 (5), 269 (80), 240 (52), 224 (32), 211 (28),181 (100), 153 (43), 69 (45)

0.7 1.0

11-O-(30-Hydroxybutanoyl)hamayne (9)b-2 3130 373 (100) 286 (25), 269 (74), 252 (25), 240 (45), 224 (38),211 (28), 181 (43), 153 (13)

2.1 8.3 14.7

A-2 (10) 3349 ?423 (8) 354 (3), 269 (18), 240 (10), 224 (33), 211 (17),181 (24), 153 (12), 69 (100)

0.4

A-3 (11) 3362 ?423 (7) 354 (3), 269 (16), 240 (11), 224 (31), 211 (18),181 (23), 153 (11), 69 (100)

0.6

A-4 (12) 3392 ?423 (6) 354 (3), 269 (18), 240 (10), 224 (30), 211 (17),181 (23), 153 (12), 69 (100)

<0.1 2.4 3.6

?3-O-(200-Butenoyl)-11-O-(30-hydroxybutanoyl)hamayne isomer (13)d

3411 441 (29) 372 (2), 269 (84), 252 (25) 240 (47), 224 (54),211 (33), 181 (86), 153 (39), 69 (100)

1.3 1.9

?3-O-(200-Butenoyl)-11-O-(30-hydroxybutanoyl)hamayne isomer (14)d

3495 441 (29) 372 (2), 269 (83), 252 (27) 240 (45), 224 (55),211 (31), 181 (85), 153 (40), 69 (100)

<0.1 3.7 4.9

3-O-(200-Butenoyl)-11-O-(30-hydroxybutanoyl)hamayne (15)b

3530 441 (29) 372 (2), 269 (84), 252 (26) 240 (47), 224 (57),211 (33), 181 (86), 153 (38), 69 (100)

4.1 10.3 26.5 35.4

3,11-O-(30 ,300-Dihydroxybutanoyl)hamayne (16)b-2

3702 459 (23) 269 (100), 252 (27), 240 (47), 224 (61),211 (39), 181 (96),153 (33)

<0.1 1.05 1.1

Total 20.8 25.8 0.8 35.6 0.2 63.6

Lycorine typeAnhydrolycorine (17)a,c-2 2502 251 (43) 250 (100), 192 (13), 191 (11), 165 (4),

164 (3), 139 (2), 124 (7)6.2 1.2 10.5 0.4 17.9 12.3

Caranine (18)a,c-3 2524 271 (56) 270 (33), 252 (46), 227 (43), 226 (100),194 (5), 154 (8)

<0.1 <0.1

Galanthine (19)a,c-4 2711 317 (20) 316 (15), 298 (9), 268 (16), 243 (89),243 (94), 242 (100), 228 (8)

0.6 1.33 <0.1

Lycorine (20)b-1 2754 287 (31) 286 (19), 268 (24), 250 (15), 227 (79),226 (100), 211 (7), 147 (15)

22.8 11.6 8.0 3.0 0.6

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Incartine (21)b-1 2761 333 (58) 332 (100), 296 (10), 259 (80), 258 (82),244 (12), 214 (6), 162 (4)

1.9 5.1 4.94 2.0 0.3

A-5 (22) 3086 ?355 (13) 269 (49), 268 (69), 250 (100), 226 (28),192 (14), 147 (15), 69 (15)

2.5 0.9

2?-O-(30-Hydroxybutanoyl)lycorine (23)d,e 3138 373 (21) 372 (16), 269 (73), 268 (100), 250 (53),227 (22), 226 (43), 147 (9)

22.1

2?-O-(30-Hydroxybutanoyl)lycorine isomer(24)d,e 3236 373 (19) 372 (15), 269 (72), 268 (100), 250 (55),227 (20), 226 (43), 147 (7)

1.7 0.4

2-O-(30-Acetoxybutanoyl)lycorine (25)b-2 3338 415 (8) 414 (4), 269 (44), 268 (58), 250 (100),227 (16), 226 (26), 192 (14)

1.6 0.9

Total 30.9 18.6 18.6 6.7 50.7 0.3 15.0

Tazettine type6-Deoxytazettine (26)a 2542 315 (21) 300 (15), 260 (5), 231 (100), 211 (15),

197 (10), 152 (8), 141 (8)2.5 <0.1 0.3

Tazettine (27)b-2 2657 331 (31) 316 (15), 298 (23), 247 (100), 230 (12),201 (15), 181 (11), 152 (7)

22.4 38.3 7.9 12.1

Macronine (28)c-5,d 2772 329 (45) 314 (64), 247 (75), 225 (13), 201 (45),139 (15), 70 (100)

<0.1 0.5 0.9

Epimacronine (29)b-4 2821 329 (27) 314 (23), 245 (100), 225 (14), 201 (83),139 (16), 70 (18)

4.5 5.4 2.63 5.3

Total 29.4 43.7 11.0 18.9

Homolycorine typeMasonine (30)c-5 2696 299 (1) 190 (2), 162 (4), 134 (1), 109 (100),

108 (25), 94 (3), 82 (3)0.2

Homolycorine (31)b-5 2767 315 (–) 206 (0.6), 178 (2), 109 (100), 150 (1),108 (22), 94 (3), 82 (3)

0.4

8-O-Demethylhomolycorine (32)b-1 2831 301 (–) 192 (0.5) 164 (2), 109 (100), 108 (25),94 (3), 82 (3)

36.3 31.8 38.6 39.5

2-Methoxyhomolycorine (33)d 2870 345 (–) 206 (0.6), 178 (2), 168 (0.5), 150 (1),139 (100), 124 (63), 94 (5)

0.57 0.8

Hippeastrine (34)b-1 2901 315 (0.1) 190 (1), 162 (4), 134 (2), 125 (100),96 (40), 82 (3)

18.8 24.3 24.3 1.7

2-Methoxy-8-O-demethylhomolycorine (35)c-6 2912 331 (–) 192 (0.2), 164 (2), 139 (100), 124 (70),94 (2), 77 (2)

1.1 2.1

Galwesine (36)c-6 2954 361 (13) 206 (3), 178 (7), 155 (90), 140 (100),112 (17), 98 (13), 85 (14)

2.3 12 4.7 <0.1

8-O-Demethylgalwesine (37)c-6 3011 347 (9) 192 (2), 164 (9), 155 (85), 140 (100),112 (18), 98 (14)

1.2 1.33 0.2 <0.1

Total 59.8 72.1 69.3 41.1

* ? is used in case of doubt in the molecular ion or the position of the substituent. More explanations are given in the text.a Comparison with a standard MS spectra from NIST 05.b Isolated standard(co-GC/MS): 1: Berkov et al. (2007b), 2: Berkov et al. (2007a), 3: Bastida et al. (1987), 4: Viladomat et al. (1990), 5: Bastida et al. (1992).c Tentative identification with MS data from literature (1: Ali et al. (1986), 2: Evidente et al. (1985), 3: Evidente et al. (1986), 4: Bastida et al. (1990), 5: Kreh et al. (1995), 6: Latvala et al. (1995)).d Tentative identification comparing with other MS spectra of known compounds.e The position of the substituent can not be solely assigned unambiguously by MS.

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The proportion of each individual compound in the alkaloid fractions was expressed as percentage of the total alkaloids(Table 1). The area of the GC/MS peaks depends not only on the concentration of the corresponding compounds but also onthe intensity of their mass spectral fragmentation. Therefore, data given in Table 1 do not express a real quantification,although they can be used to compare the samples, which was the objective of this work.

2.4. TLC analysis

Leaves from 50 individuals per each population were dried at 50 �C, then powdered and analyzed by TLC as described inBerkov et al. (2004).

3. Results and discussions

3.1. Alkaloid identification

A great number of alkaloids that are in the amaryllidaceous plant extracts has been separated effectively and identifiedvery quickly by GC/MS (Kreh et al., 1995; Berkov et al., 2004) indicating that this method for chemical analysis is usefuland reliable for studies on the alkaloid metabolism in this family.

Thirty-seven compounds with mass spectral characteristics of Amaryllidaceae alkaloids were detected in the plant organsof the studied species (Table 1). Twenty-five of them were found in G. nivalis and 17 in G. elwesii. Only five of these alkaloidswere found in both species, namely compounds 2, 4, 17, 20 and 21. Many of the identified alkaloids such as 5, 6, 7, 20, 27, 31,and 34 possess pharmacological and biological activity (Campbell et al., 2000; Bastida et al., 2006).

Compound 8 has an intensive ion at m/z 355 and mass spectral fragmentation very similar to previously isolated hamaynederivatives 9, 15, and 16 (Berkov et al., 2007a) indicating a substitution of the hydroxyl group at C-11 of hamayne. The elim-ination of 69 mass units (ion fragments at m/z 286 and 69) indicates presence of a 2-butenoyl group. Similarly, compounds10–12 showed intensive ions at m/z 423, indicating two 2-butenoyl moieties, at C-3 and C-11, respectively. The presence of 2-butenoyl moieties, however, could be a result of elimination of a hydroxybutanoyl group under GC/MS condition as found in3,11,30-O-(30,300,3%-trihydroxybutanoyl)hamayne, a compound which does not show a molecular ion (Berkov et al., 2007a).Unambiguous identification of compounds 8, 10–12 could be done after their isolation and NMR examination.

Alkaloids 22–24 showed mass spectral fragmentation characteristic for lycorine type derivatives. Their mass spectra werevery similar to those of 2-O-(30-acetoxybutanoyl)lycorine (25) (Berkov et al., 2007a). However, compounds 23 and 24 showedidentical mass spectral fragmentation and adjacent peaks, like isomers (Kreh et al., 1995), and Mþ at m/z 373, thus indicatinga substitution with a 3-hydroxybutanoyl group most probably at C-2, similarly to 25. Compound 22 showed an Mþ at m/z 355,as well as an intensive ion at m/z 69 indicating elimination of a 2-butenoyl group. Similarly to compounds 8, 10–12, thecomplete identification of 22–24 is possible after their isolation.

Compound 28, tentatively identified as macronine, showed a mass spectral fragmentation similar to that of 29 (Viladomatet al., 1990), but the ion fragment at m/z 70 was considerably more intensive (base ion) as compared to the same fragment of29, thus indicating that the methoxyl group at C-3 is in a-position as in criwelline (Duffield et al., 1965).

With the exception of galwesine (36) and 8-O-demethylgalwesine (37), the rest of homolycorine type alkaloids showedweak molecular ions. A general feature of the homolycorine type alkaloids with a double bound D3,4 is the low intensityof their molecular ions (Kreh et al., 1995). These alkaloids have intensive fragments in the low mass range representingthe pyrrolidine ring fragments, and less intensive ion fragments in the middle mass range which encompasses the aromaticlactone or hemiacetal moiety (Jeffs et al., 1985). Compound 30 showed a base ion at m/z 109 indicating that there is no sub-stitution at C-2, and less intensive fragments at m/z 162 and 190 suggesting a lactone moiety with a methylenedioxy group atC-8/C-9. The GC/MS spectrum and RI of 30 were congruent with those reported for masonine by Kreh et al. (1995). Compound35, detected in G. elwesii, showed intensive ion fragments at m/z 124 and 139 thus indicating a methoxyl group at C-2, and lessintensive ion fragments at m/z 164 and 192 characteristic for a lactone moiety with one methoxyl and hydroxyl groups at C-8and C-9, respectively. The position of the methoxyl group cannot be ascribed by MS solely. The mass spectrum of 35 wascongruent with that of 2-methoxy-9-O-demethylhomolycorine, an alkaloid isolated from G. elwesii (Latvala et al., 1995).Compound 33 also showed intensive ion fragments at m/z 124 and 139 and less intensive ion fragments at m/z 178 and206 thus indicating a lactone moiety with two methoxyl groups at C-8 and C-9. This compound was tentatively identifiedas 2-methoxyhomolycorine.

It is interesting to emphasize that the compounds 8, 10–14, 22 and 23 had not been previously described and theirisolation could provide additional information on their structure and bioactivity. As well, compounds 3, 7, 18, 21 and 26are reported for the first time in plants of the genus Galanthus.

3.2. Distribution of alkaloids in plant organs

Considering the organ-to-organ distribution of the alkaloids, each plant organ displayed different quantitative andqualitative alkaloid profile (Table 1).

The predominant alkaloids in G. elwesii roots, bulbs and leaves were those of homolycorine type, whereas flowers ac-cumulated mainly tyramine type compounds. The number of alkaloids present in the flowers was considerably lower to

S. Berkov et al. / Biochemical Systematics and Ecology 36 (2008) 638–645 643

that of the other plant organs. In the roots, bulbs and leaves, the most abundant alkaloids were 8-O-demethylhomolycor-ine (32) and hippeastrine (34) whereas in the flowers it was hordenine (2). In this plant species, the lycorine type alka-loids decreased quantitatively and qualitatively from roots (18.6%, four compounds) to bulbs (6.7%, three compounds) andleaves (0.3%, two compounds), and no alkaloids of this type were detected in flowers. In contrast, the hordenine contentincreased from the underground to the aerial parts (up to 58.9% of the alkaloid mixture in flowers). It is noteworthy thatthe high accumulation of this protoalkaloid, which is not typical for plants of the Amaryllidaceae family. Hordenine hasbeen found mainly in other plant families like Poaceae (Hordeum vulgare), Cactaceae (in a very wide range of species), andin a few algae and fungi. It possesses diuretic, disinfectant and antihypotensive properties, and also acts as feeding repel-lent for grasshoppers (DNP). Considering the high accumulation of hordenine in the aerial parts, probably its biological

NR4O

R3O

R2

H

H

R1

O

MeN

O

R2O

R3O

R1

H

Tyramine type1 R1=CH3, R2=H2* Rl=R2=CH3

N17*

18 R1=H, R2=OH, R3+R4=CH219 R1=OCH3, R2=OH, R3=R4=CH320* R1=R2=OH, R3+R4=CH22223, 24 R1=OCOCH2CHOHCH3, R2=OH, R3+R4=CH2

25 R1=OCOCH2CHCH3, R2=OH, R3+R4=CH2

N

HO

H

H

OH

O

O

OO

O21*

OCOCH3

Lycorine type

30 R1=H, R2+R3=CH231 R1=H, R2=R3=CH332 R1=R3=H, R2=CH333 R1=OCH3,R2=R3=CH334 R1=OH, R2+R3=CH235 R1=R2=OCH3, R3=H

O

MeN

O

R1O

R2O

OCH3

H O

36 R1=R2=CH337 R1=CH3, R2=H

OH

NR1 R2

NHHO

H3CO

Tazettine type26 R1=OCH3, R2=R3=R4=R5=H27 R1=OCH3, R2=R4=R5=H, R3=OH28 R1=OCH3, R2=R3=H, R4+R5=O29 R1=R3=H, R2=OCH3, R4+R5=O

O

NCH3

R3

H

O

O

R2R1

R4R5

HO

HO CHO

HO

H2N

O-Methylnorbelladine

TyramineProtocatechuic aldehyde

Haemanthamine type5 R1=OH, R2=R3=H6 R1=OCH3, R2=H, R3=OH7 R1=H, R2=OH, R3=OH89 R1=H, R2=OH, R3= OCOCH2CHOHCH310, 11, 1213, 14, 15 R1=H, R2=OCOCHCHCH3,

R3=OCOCH2CHOHCH316 R1=H, R2=R3=OCOCH2CHOHCH3

N

R3

H

O

O

R2

R1

NHCH3O

O

N

O

O

3

4*

L-Tyr

L-Phe

ortho-para'

para-para'

Homolycorine type

O-NorbelladineOH

OH

Fig. 1. Structures and biosynthetic relationships of the alkaloids identified in G. nivalis (- - - -) and G. elwesii (ddd) (after Bastida et al., 2006). *Compounds foundin both species.

S. Berkov et al. / Biochemical Systematics and Ecology 36 (2008) 638–645644

activity is related to the plant defense against herbivores (snowdrops are the earliest plants flowering at the end ofwinter).

The predominant alkaloids in G. nivalis roots were found to belong to the lycorine (30.9%) and tazettine (29.4%) structuraltypes (Table 1). Bulbs were dominated by tazettine (43.7%), leaves by lycorine (50.7%) and flowers by haemanthamine (63.6%)type compounds. The number of alkaloids in the aerial parts (leaves and flowers) was considerably higher comparing it withthe underground parts of the plant (bulbs and roots). The main alkaloids in roots were lycorine (20, 22.8%) and tazettine (27,22.4%). Tazettine was the main alkaloid in the bulbs (38.3%), whereas 3-O-(200-butenoyl)-11-O-(30-hydroxybutanoyl)hamayne(15) was the dominant compound in the aerial parts, representing 26.5 and 35.4% of the total alkaloid fraction in leaves andflowers, respectively. Tazettine was found in a relatively low concentration in the aerial parts. It is also noteworthy that theoccurrence of hamayne and lycorine derivatives with 2-butenoyl and 3-hydroxybutanoyl groups. The quantity and number ofthese derivatives increased from the underground to the aerial parts of the plants. Thus, hamayne derivatives represent 6.3,18.6, 35.6 and 63.6% of the alkaloid mixture in roots (three compounds), bulbs (four compounds), leaves (six compounds) andflowers (nine compounds), respectively. Lycorine derivatives were detected neither in roots nor in bulbs, but they wereaccumulated in leaves (four compounds, 25.9%) and flowers (three compounds, 2.2%). No free hamayne was detected inthe aerial parts whereas lycorine represents only 3 and 0.6% in leaves and flowers, respectively. It is well known that lycorinehas a wide range of biological activities including emetic, cytotoxic, antiviral, insecticidal, etc. (Bastida et al., 2006). On theother hand, hamayne has been found to exhibit AChE inhibitory activity and cytotoxic effects (Campbell et al., 2000;Houghton et al., 2004). Probably, 2-butenoyl and 3-hydroxybutanoyl derivatives of lycorine and hamayne are biologicallyless active than their free bases and can be accumulated in the aerial parts. Related to this, Ghosal et al. (1990) reportedthe rapid hydrolysis of lycorine conjugates after injury or animal attack, thus suggesting that these compounds are involvedin mechanisms of plant defense.

The intrapopulation variability of the alkaloid profiles was studied by TLC having used 50 individuals of each population. G.nivalis and G. elwesii showed different leaf alkaloid profiles and no variation within populations was observed. Plants withintermediate alkaloid patterns were not found, which suggests that there is no hybridization between these two species. Ifthere was, it would take place at a very low rate (undetected in our study).

The effect of environmental factors (temperature, soil composition, etc.) on the alkaloid patterns of these sympatricspecies can be neglected and the differences between them must be determined by their genetic background. As mentionedabove, only five alkaloids out of 37 compounds were found in both species (Table 1). It is noteworthy that the biochemicaldifferentiation of the two studied species has led to synthesis of tazettine type alkaloids in G. nivalis and that of homolycorinetype in G. elwesii, which are biosynthetically related with the more general structural types, haemanthamine and lycorine(both synthesized by these two plant species). The alkaloid pattern of G. nivalis was dominated by compounds comingfrom a para–para0 oxidative coupling of O-methylnorbelladine (haemanthamine and tazettine type alkaloids, Fig. 1). The con-jugated and free lycorine type alkaloids coming from an ortho–para0 oxidative coupling were less abundant, detected mainlyin the underground parts. Homolycorine type alkaloids were not detected in this plant species. In contrast to G. nivalis, thealkaloid pattern of G. elwesii was dominated mainly by compounds coming from ortho–para0 oxidative coupling: free lycorinetype alkaloids and those of homolycorine type. Thus, G. elwesii did not synthesize (or did it in trace amounts) ismine (3) andtrisphaeridine (4). The synthesis of para–para0 oxidative products in G. elwesii was very weak (only haemanthamine type al-kaloids, up to 0.8% of the total alkaloid production, two compounds). All these facts indicate a deep difference in the second-ary metabolism between these two sympatric populations (species) and gave a chemotaxonomical support for the division ofG. nivalis and G. elwesii into different taxons.

The use of secondary metabolites, and Amaryllidaceae alkaloids in particular, as chemotaxonomic markers should be veryprecocious because of the variations found in the alkaloid patterns between the populations. Tazettine and haemanthaminetype alkaloids, as well as galanthamine type alkaloids (the last not found in these populations), have been reported for otherpopulations of G. elwesii (Latvala et al., 1995; Berkov et al., 2004).

Acknowledgements

This work was partially financed by the Generalitat de Catalunya (2005 SGR-00020). S. Berkov thanks the Spanish Minis-terio de Educacion y Ciencia for a research fellowship (SB2004-0062). The authors also thank Dr. Asuncion Marın, ServeisCientificotecnics, Universitat de Barcelona (Facultat de Farmacia), for performing the GC/MS analyses.

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